Histone demethylase KDM6A directly senses oxygen to control chromatin and cell fate

Abstract

Oxygen sensing is central to metazoan biology and has implications for human disease. Mammalian cells express multiple oxygen-dependent enzymes called 2-oxoglutarate (OG)-dependent dioxygenases (2-OGDDs), but they vary in their oxygen affinities and hence their ability to sense oxygen. The 2-OGDD histone demethylases control histone methylation. Hypoxia increases histone methylation, but whether this reflects direct effects on histone demethylases or indirect effects caused by the hypoxic induction of the HIF (hypoxia-inducible factor) transcription factor or the 2-OG antagonist 2-hydroxyglutarate (2-HG) is unclear. Here, we report that hypoxia promotes histone methylation in a HIF- and 2-HG-independent manner. We found that the H3K27 histone demethylase KDM6A/UTX, but not its paralog KDM6B, is oxygen sensitive. KDM6A loss, like hypoxia, prevented H3K27 demethylation and blocked cellular differentiation. Restoring H3K27 methylation homeostasis in hypoxic cells reversed these effects. Thus, oxygen directly affects chromatin regulators to control cell fate.

Fig. 1.. Hypoxia Causes HIF-Independent Histone Hypermethylation.

(A-C) Vector schematic (A), Histone modification profiling by mass spectrometry (B), and Histone immunoblot analysis (C) of Arnt-deficient mouse Hepatoma (mHepa-1 c4) cells that were lentivirally transduced to produce the indicated V5-tagged proteins and cultured at the indicated oxygen levels for 4 days. In (B), rows represent two biological replicates of the indicated samples and the color in each cell represents log₂ fold change relative to all samples in the column, normalized for total histone using an internal control peptide (Histone H3: residues 41-49). (D-F) Coomassie blue staining (D) and biochemical analysis of baculovirally expressed and purified JumonjiC (JmjC) catalytic domains of KDM6A [KDM6A*] (E) and KDM6B [KDM6B*] (F). K_M values are mean +/− SD (N=3). (G) Immunohistochemical analysis of the indicated tissues derived from representative male and female age-matched mice

Fig. 2.. Regulation of Myogenic Differentiation by the KDM6A H3K27 Demethylase.

(A-B) Immunofluorescence analysis of C2C12 cells that were lentivirally transduced to express the shRNAs targeting Kdm6A (A) or Kdm6B (B) and then cultured in differentiation media for 4 days. (C-E) Immunoblot analysis of C2C12 cells lentivirally transduced to express the indicated sgRNAs and cultured for 4 days either in growth media (GM) or differentiation media (DM), as indicated. (E) Immunoblot analysis of C2C12 cells expressing, where indicated, Kdm6a sg2 [described in (C) and (D)] that were lentivirally transduced to produce either GFP (control) or wild-type human KDM6A [6A(WT)] and then cultured in DM at the indicated oxygen concentrations for 4 days. The mouse Kdm6A sg2 target sequence is not conserved in human KDM6A. (F) Immunoblot analysis of histones from C2C12 cells expressing the indicated sgRNA that were cultured at the indicated oxygen concentrations for 3 days. (G-J) Heatmap representing mRNA levels determined by RNA-Seq (G) and H3K27me3 levels determined by ChIP-Seq analysis at the Actc1 (H), Myl1 (I), and the Myog (J) loci from two biological replicates (A and B) of C2C12 cells cultured in the indicated media for 4 days at the indicated oxygen concentration.

Fig. 3.. Restoring the Balance of H3K27 Methyltransferase Activity to H3K27 Demethylase Activity Rescues Myogenic Differentiation Under Hypoxic Conditions.

(A) Model for control of H3K27 methylation by the indicated opposing demethylases (“erasers”) and methyltransferases (“writers”). (B) Immunoblot analysis of C2C12 cells lentivirally transduced to express the indicated sgRNAs and cultured under the indicated conditions. (C) Structural models of the KDM6A (pink) and KDM6B (cyan) catalytic pockets. The non-conserved M¹¹⁹⁰ (KDM6A) → T¹⁴³⁴ (KDM6B) and the E¹³³⁵ (KDM6A) → D¹⁵⁷⁹ (KDM6B) are highlighted. Peptidic H3K27me3 substrate (yellow), Fe⁺² (orange), 2-oxoglutarate (green), and Zn⁺² (grey) are shown. (D-E) Michaelis-Menten plots (inset Lineweaver-Burk plot) and measured oxygen K_M and V_max values (mean +/− SD, N=3) of recombinant KDM6A wild-type and the MT/ED mutant. (F-G) Real-Time qPCR analysis (mean +/− SD, N =3) of the indicated genes (F) and immunofluorescence analysis (G) of C2C12 cells transduced to produce wild-type human KDM6A or the KDM6A MT/ED variant and then cultured in DM at the indicated oxygen concentrations for 4 days.